Dscam is an axon guidance receptor that is required for the formation of neuronal connections in the Drosophila nervous system. Dscam loss-of-function phenotypes have been characterized in detail for an embryonic nerve called Bolwig's nerve (BN). Phenotypic studies of Dscam mutants suggest that Dscam protein is critical for mediating a short-range interaction between BN and an intermediate target. Dscam also is required for the specification of other connections in the developing embryo. Dscam protein contains 10 Ig repeats, 6 fibronectin type III repeats, a single transmembrane segment and a cytoplasmic tail with multiple binding sites for the Dock adapter protein, a signaling component connecting Dscam to actin cytoskeletal regulators. Alternative splicing of Dscam may lead to more than 38,000 forms of the receptor. These receptors share the same domain structure, but differ in amino acid sequence in 4 different domains. Three of the Ig domains and the transmembrane domain are variable. This variability reflects the use of alternative exons. In this proposal, the specific form of Dscam expressed in BN will be determined using RT-PCR. Transgene rescue experiments will be used to verify that this form is functional in BN. Transgenes containing different variable domains will be tested for function by targeted expression in BN. Additional experiments will be directed towards determining the pattern of expression of Dscam isoforms in the developing embryo and the developing eye imaginal disc using both in situ hybridization and RT-PCR techniques. These studies will form the foundation for determining whether different Dscam isoforms contribute to a molecular code for axon guidance and targeting. As Drosophila Dscam is structurally related to a human gene implicated in Down Syndrome, the studies described in this grant may provide insights into the abnormalities in brain development in Down Syndrome patients.